CN107130011A - A kind of microscopic observation method of food-borne pathogens single cells grown process - Google Patents
A kind of microscopic observation method of food-borne pathogens single cells grown process Download PDFInfo
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Abstract
The present invention provides a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens, covering adapts to the solid medium or double layer culture base of food-borne pathogenic bacteria growing i.e. on food-borne pathogens, is then observed the unicellular individual growth process of food-borne pathogens with microscope again；Described covering double layer culture base, i.e., from top to bottom, solid medium, fluid nutrient medium successively.This method is applied to the observation of the unicellular individual process of all food-borne pathogens, and uninterruptedly can directly observe the fission process of cell individual.This method is to secure food-borne pathogens, while avoid causes the problem of growing environment subalimentation and solid medium are dehydrated because long-time is observed.
Description
Technical field
The present invention relates to the microscopic observation method of individual cell level food-borne pathogens, it is more particularly to a kind of based on solid or
The microscopic observation method of the unicellular individual growth process of food-borne pathogens of double layer culture base.
Background technology
Common food-borne pathogens have salmonella, vibrio parahemolyticus, single increasing listeria spp, E
Bacterium, staphylococcus aureus, Bacillus cereus etc..In recent years, both at home and abroad by the microbial food poisoning thing of food-borne pathogenic
Part increasingly gets more and more people's extensive concerning.And most food poisonings be as caused by low bacterium amount contaminated food products, so
In food-borne pathogens growth rhythm research, the unicellular individual research of food-borne pathogens seems particularly important.
In current food-borne pathogens individual cell level research, observation procedure is particularly significant, but existing observation procedure is still
There are many deficiencies.The conventional method for probing into growth rhythm under microbial single-cell level has turbidimetry and microscopic observation method.Its
The Testing index of middle turbidimetry is the turbidity of bacteria suspension, it is impossible to be directly observed growth and the fission process of cell individual.And
The side of the unicellular individual true growth fission process of food-borne pathogens is studied from individual cell level based on light microscope
Method still suffers from some problems.Such as, the agar film being inoculated with is placed in micro- Microscopic observation ferment of regularly taking pictures by Kelly etc. [1]
The process of mother cell division, wherein agar thin slice can prevent yeast cells disordered motion, regularly take pictures, and can record yeast list
The fission process of cell individual.But in long-time observation process, cell individual can stop due to growing environment subalimentation
Only growth or dead, and then the Continuous Observation that influence grows to it.Meanwhile, increase over time, agar thin slice is also easy to produce dehydration
Phenomenon.When it is liquid [2] to observe matrix, the nutrient solution that the daughter cell of division is flowed is taken away, and causes nothing in observation process
Method observes the unicellular individual whole growth fission process of food-borne pathogens.
Therefore, suitable method individual unicellular to food-borne pathogens not yet carries out observation work at present.
Bibliography：
[1]、Kelly C D, Rahn O. The Growth Rate of Individual Bacterial Cells [J].
Journal of Bacteriology, 1932, 23(2): 147-153
[2]、Elfwing A, Lemarc Y, Baranyi J, et al. Observing Growth and Division
of Large Numbers of Individual Bacteria by Image Analysis [J]. Applied and
Environmental Microbiology, 2004, 70(2): 675-678。
The content of the invention
, should it is an object of the invention to provide a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens
Observation procedure has selected the solid medium or double layer culture base suitable for all food-borne pathogens, therefore this method is suitable
For the observation of the unicellular individual process of all food-borne pathogens, and it uninterruptedly can directly observe the division of cell individual
Journey.During from solid medium as the individual mounting medium of food-borne pathogens, it can avoid having food-borne pathogenic amphitrichous
The problem of bacterium individual freedom is moved.During with solid-liquid Double-Medium, this method is to secure bacterium, at the same avoid because it is long when
Between observation and cause the problem of growing environment subalimentation and solid medium are dehydrated.
Technical scheme
A kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens, i.e., cover suitable on food-borne pathogens
The solid medium or double layer culture base of food-borne pathogenic bacteria growing are answered, food-borne cause is then observed with microscope again
The unicellular individual growth process of germ；
From top to bottom described covering double layer culture base, i.e., be followed successively by solid medium, fluid nutrient medium；
When covering adapts to the solid medium of food-borne pathogenic bacteria growing on food-borne pathogens, using just putting type or inversion
Type microscope is observed；
It is aobvious using inversion type when covering adapts to the double layer culture base of food-borne pathogenic bacteria growing on food-borne pathogens
Micro mirror is observed.
A kind of microscopic observation method of the unicellular individual growth process of above-mentioned food-borne pathogens, specifically includes following step
Suddenly：
（1）, culture medium preparation
Only with Listeria monocytogenes in embodiments of the invention（Listeria monocytogenes
ATCC13932, hereinafter referred to as Listeria monocytogenes, Shanghai University of Science and Technology professor Liu Qing give（Donor address：Shanghai City Yangpu
The Shanghai University of Science and Technology of area military project road 516, contact method：13916919896）Exemplified by illustrate, but be not intended to limit this observation
Method other food-borne pathogens use, when simply using other food-borne pathogens, to adapting to the food-borne pathogens
The solid medium or double layer culture base of growth will do the adjustment of adaptability；
The described adaptation food-borne pathogens are Listeria monocytogenesListeria monocytogenesATCC13932 gives birth to
Long solid medium is TSA-YE culture mediums, its raw material composition, by every liter of calculating, by 17g tryptones, 3g multivalence peptone, 6g
Yeast extract, 5g sodium chloride, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, the water composition of 15g agar and surplus；
Above-mentioned TSA-YE culture mediums are by comprising the following steps that method is prepared from：
With reference to GB 4789.30-2016：Tryptone, multivalence peptone, yeast extract, sodium chloride, phosphoric acid hydrogen are sequentially added in a reservoir
Dipotassium, glucose and agar, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved, and pH is to 7.2 ± 0.2 for regulation,
Then packing, 121 DEG C of autoclaving 15min are put in standby in 50 DEG C of insulating box；
Described adaptation food-borne pathogens are that the fluid nutrient medium of Listeria monocytogenes growth is TSB-YE culture mediums, and it is former
Material composition, by every liter calculating, by 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g sodium chloride, 2.5g dipotassium hydrogen phosphates,
2.5g glucose and the water of surplus composition；
Above-mentioned TSB-YE culture mediums are by comprising the following steps that method is prepared from：
With reference to GB 4789.30-2016：Tryptone, multivalence peptone, yeast extract, sodium chloride, phosphoric acid hydrogen are sequentially added in a reservoir
Dipotassium and glucose, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved, with regulation pH to 7.2 ± 0.2, point
Then dress, 121 DEG C of autoclaving 15min are put in superclean bench standby；
（2）, unicellular Listeria monocytogenes bacteria suspension after dilution preparation
Take and increase the Listeria monocytogenes to be measured after bacteriumListeria monocytogenesATCC13932 bacteria suspensions, it is used and walked
Suddenly（1）The TSB-YE culture mediums prepared are diluted to Listeria monocytogenesListeria monocytogenes
ATCC13932 cell concentrations reach that (cfu (Colony-Forming Units, CFU), is produced dilute 3cfu/mL
Listeria monocytogenes after releasingListeria monocytogenesATCC13932 bacteria suspensions, wherein cfu/mL refer to every milli
Rise the Listeria monocytogenes after dilutionListeria monocytogenes The single increasing Li Si contained in ATCC13932 bacteria suspensions
Special bacteriumListeria monocytogenesATCC13932 total plate count；
（3）, being used for based on solid medium or double layer culture base observe Listeria monocytogenesListeria monocytogenesThe preparation of the sample of the unicellular individual growth processes of ATCC13932
First, 20 μ L steps are taken（2）Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacterium
Suspension is into sterile 35mm Glass bottom culture dish, and slow shaking by swirling, makes the Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacteria suspensions are laid in the bottom of Glass bottom culture dish, stand 5min；
Then 100 μ L steps are drawn（1）After TSA-YE culture mediums after sterilizing are slightly cooled down, it is added to after being diluted in Glass bottom culture dish
Listeria monocytogenesListeria monocytogenesOn ATCC13932 bacteria suspensions, stand until TSA-YE culture mediums are complete
Full solidification, then covers the upper lid of Glass bottom culture dish, and produce is used to observe Listeria monocytogenes based on solid mediumListeria monocytogenesThe sample of the unicellular individual growth processes of ATCC13932；
Or 100 μ L steps are drawn first（1）After TSA-YE culture mediums after sterilizing are slightly cooled down, it is added to dilute in Glass bottom culture dish
Listeria monocytogenes after releasingListeria monocytogenesOn ATCC13932 bacteria suspensions, stand until TSA-YE cultures
Base solidifies completely, then draws 100-200 μ L steps（1）TSB-YE culture mediums after sterilizing, are covered in the TSA- after above-mentioned solidification
On YE culture mediums, the upper lid of Glass bottom culture dish is then covered, produces and Lee is increased for observing list based on double layer culture base
This special bacteriumListeria monocytogenesThe sample of the unicellular individual growth processes of ATCC13932；
（4）, microscope is observed
Using type or the microscopical 100 times of object lens of inversion type are just put, the single increasing corresponded in the bottom of Glass bottom culture dish after dilution
ListeriaListeria monocytogenesAt the observation window of ATCC13932 bacteria suspensions, by adjusting resolution ratio, the visual field
Brightness, focal length, find step（3）Being used for based on solid medium of gained observes Listeria monocytogenesListeria monocytogenesUnicellular individual in the sample of the unicellular individual growth processes of ATCC13932, is then observed list
Increase ListeriaListeria monocytogenesThe single celled fission processes of ATCC13932 and cell division anaplasia at any time
The process of change；
Or the microscopical 100 times of object lens of inversion type are used, the single increasing Liszt corresponded in the bottom of Glass bottom culture dish after dilution
BacteriumListeria monocytogenesAt the observation window of ATCC13932 bacteria suspensions, by adjusting resolution ratio, field luminance, Jiao
Away from finding step（3）Being used for based on double layer culture base of gained observes Listeria monocytogenesListeria monocytogenesUnicellular individual in the sample of the unicellular individual growth processes of ATCC13932, is then observed list
Increase ListeriaListeria monocytogenesThe single celled fission processes of ATCC13932 and cell division anaplasia at any time
The process of change.
The advantageous effects of the present invention
It is slender that a kind of being used for based on solid medium or based on double layer culture base of the present invention observes food-borne pathogens
The method of born of the same parents' individual growth process, due to having selected the solid medium or solid-liquid that are applied to food-borne pathogens to be observed double
The overall process that layer culture medium is observed thalline carries out nutrition supply, therefore the unicellular individual growth process of food-borne pathogens is entered
During row observation, the fission process of cell individual uninterruptedly can be directly observed.
Further, being used for based on solid medium of the invention observes the unicellular individual growth process of food-borne pathogens
Method, due to from solid medium as food-borne pathogens individual mounting medium when, therefore can avoid with flagellum
Food-borne pathogens individual freedom the problem of move.Also, the solid medium in the present invention is to be adapted to food-borne pathogenic
The solid medium of bacteria growing, in observation process, can provide nutriment for food-borne pathogens.
Further, being used for based on double layer culture base observes the side of the unicellular individual growth process of food-borne pathogens
Method, due to the use of solid-liquid Double-Medium, this method is to secure bacterium, while avoid causes because long-time is observed
The problem of growing environment subalimentation and solid medium are dehydrated.
Further, solid medium of the invention or being used for based on double layer culture base observe food-borne pathogens list
The method of cell individual growth course, can be according to the type of microscopic system（Just putting type or inversion type microscopic system）To select
Required observation system, observation of the solid monolayer cultivation matrix system suitable for just putting type and inversion type microscopic system, and solid-liquid
The long-time that Double-Medium system is suitable to inversion type microscopic system is observed.
Brief description of the drawings
1st, being used for based on double layer culture base observes Listeria monocytogenesListeria monocytogenes
The structural representation of the sample of the unicellular individual growth processes of ATCC13932；
2nd, the Listeria monocytogenes based on double layer culture baseListeria monocytogenesATCC13932 is unicellular to be split
The growth course schematic diagram of change；
3rd, using the time as abscissa, drawn by ordinate of the cell number after fission, the Listeria monocytogenes of gainedListeria monocytogenesCell number after the unicellular fissions of ATCC13932 changes over time the schematic diagram of situation.
Embodiment
The present invention is further elaborated below by embodiment, but is not intended to limit this method in other similar observation systems
Application in system.
Only increase listeria spp with single in following embodimentListeria monocytogenesATCC13932, Shanghai
Polytechnics professor Liu Qing gives（Donor address：No. 516, Military Road, Yangpu District,Shanghai City Shanghai University of Science and Technology, contact method：
13916919896）Illustrated for citing, but be not intended to limit this method in the unicellular individual growth mistake of other food-borne pathogens
Application in journey observation.
Type microscope of just putting used is BX41TF-5 type biomicroscopes in embodiment, is purchased from Japanese Olympus strain
Formula commercial firm；
Inversion type microscope is TS100 inversion type biomicroscopes, is purchased from Japanese Olympus Co., Ltd..
Tryptone used, multivalence peptone, yeast extract, sodium chloride, dipotassium hydrogen phosphate, glucose in various embodiments of the present invention
It is Chemical Reagent Co., Ltd., Sinopharm Group's production with agar.
Embodiment 1
A kind of food-borne pathogens are Listeria monocytogenesListeria monocytogenesThe unicellular individuals of ATCC13932
The microscopic observation method of growth course, i.e., singly increasing listeria sppListeria monocytogenesATCC13932 overlyings
Lid adapts to Listeria monocytogenesListeria monocytogenesThe double layer culture base of ATCC13932 growths, is then adopted
Listeria monocytogenes are observed with inversion type microscopeListeria monocytogenesThe unicellular individuals of ATCC13932
Growth course；
Described applicable Listeria monocytogenesListeria monocytogenesThe double layer culture of ATCC13932 growths
From top to bottom base, i.e., be followed successively by solid medium, fluid nutrient medium.
Described solid medium is TSA-YE culture mediums, its raw material composition, by every liter of calculating, by 17g tryptones, 3g
Multivalence peptone, 6g yeast extracts, 5g sodium chloride, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, the water composition of 15g agar and surplus；
Above-mentioned TSA-YE culture mediums are by comprising the following steps that method is prepared from：
With reference to GB 4789.30-2016：Tryptone, multivalence peptone, yeast extract, sodium chloride, phosphoric acid hydrogen are sequentially added in a reservoir
Dipotassium, glucose and agar, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved, and pH is to 7.2 ± 0.2 for regulation,
Packing, 121 DEG C of autoclaving 15min are standby；
Described fluid nutrient medium is TSB-YE culture mediums, and its raw material composition is more by 17g tryptones, 3g by by every liter of calculating
Valency peptone, 6g yeast extracts, 5g sodium chloride, 2.5g dipotassium hydrogen phosphates, the water composition of 2.5g glucose and surplus；
Above-mentioned TSB-YE culture mediums are by comprising the following steps that method is prepared from：
With reference to GB 4789.30-2016：Tryptone, multivalence peptone, yeast extract, sodium chloride, phosphoric acid hydrogen are sequentially added in a reservoir
Dipotassium and glucose, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved, with regulation pH to 7.2 ± 0.2, point
Dress, 121 DEG C of autoclaving 15min are standby.
A kind of above-mentioned Listeria monocytogenesListeria monocytogenesThe unicellular individual growths of ATCC13932
The microscopic observation method of process, specifically includes following steps：
（1）, TSA-YE culture mediums, the preparation of TSB-YE culture mediums
1., the preparation of TSA-YE culture mediums
It is to add 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g chlorinations successively in a reservoir with reference to GB 4789.30-2016
Sodium, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, 15g agar, then add water and are settled to 1 liter, then heating stirring to completely it is molten
Solution, adjusts pH to 7.2 ± 0.2, and packing, 121 DEG C of autoclaving 15min produce TSA-YE culture mediums；
2., the preparation of TSB-YE culture mediums
Sequentially add 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g chlorinations in a reservoir with reference to GB 4789.30-2016
Sodium, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, then add water and are settled to 1 liter, and then heating stirring adjusts pH to being completely dissolved
To 7.2 ± 0.2, packing, 121 DEG C of autoclaving 15min produce TSB-YE culture mediums, are then put in standby on superclean bench
With；
（2）, Listeria monocytogenes after dilutionListeria monocytogenesThe preparation of ATCC13932 bacteria suspensions
Take and increase the Listeria monocytogenes to be measured after bacteriumListeria monocytogenesATCC13932 bacteria suspensions, it is used and walked
Suddenly（1）The TSB-YE culture mediums prepared are diluted to Listeria monocytogenesListeria monocytogenes
ATCC13932 cell concentrations reach that (cfu (Colony-Forming Units, CFU), is produced dilute 3cfu/mL
Listeria monocytogenes after releasingListeria monocytogenesATCC13932 suspensions, wherein cfu/mL refer to every milliliter
Listeria monocytogenes after dilutionListeria monocytogenesThe single increasing Liszt contained in ATCC13932 bacteria suspensions
BacteriumListeria monocytogenesATCC13932 total plate count；
（3）, based on double layer culture base be used for observe Listeria monocytogenesListeria monocytogenes
The preparation of the sample of the unicellular individual growth processes of ATCC13932, being used for based on double layer culture base observes list
Increase ListeriaListeria monocytogenesThe structural representation of the sample of the unicellular individual growth processes of ATCC13932
Figure as shown in figure 1, wherein 1 be Glass bottom culture dish, 2 be the upper lid of Glass bottom culture dish, 3 be dilution after Listeria monocytogenesListeria monocytogenesATCC13932 bacteria suspensions, 4 be TSA-YE culture mediums, 5 be TSB-YE culture mediums, it is prepared
Process is comprised the following steps that；
First, 20 μ L steps are taken（2）Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacterium
Suspension 3 is into sterile 35mm Glass bottom culture dish 1, and slow shaking by swirling, makes the Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacteria suspensions 3 are laid in the bottom of Glass bottom culture dish 1, stand 5min；
Then 100 μ L steps are drawn（1）After TSA-YE culture mediums 4 after sterilizing are slightly cooled down, it is added in Glass bottom culture dish and dilutes
Listeria monocytogenes afterwardsListeria monocytogenesOn ATCC13932 bacteria suspensions 3, stand until TSA-YE culture mediums
4 solidifications completely；
Then 100-200 μ L steps are drawn again（1）TSB-YE culture mediums 5 after sterilizing, are covered in the TSA-YE after above-mentioned solidification
On culture medium 4, the upper lid 2 of Glass bottom culture dish is then covered, produces and Lee is increased for observing list based on double layer culture base
This special bacteriumListeria monocytogenesThe sample of the unicellular individual growth processes of ATCC13932；
（4）, microscope is observed
Using the microscopical 100 times of object lens of inversion type, the Listeria monocytogenes after corresponding to dilution in the bottom of Glass bottom culture dish 2Listeria monocytogenesAt the observation window of ATCC13932 bacteria suspensions 3, by adjusting resolution ratio, field luminance, Jiao
Away from finding step（3）Being used for based on double layer culture base of gained observes Listeria monocytogenesListeria monocytogenesUnicellular individual in the sample of the unicellular individual growth processes of ATCC13932, is then observed, and sees
Survey process record Listeria monocytogenesListeria monocytogenesThe single celled fission processes of ATCC13932 and cell point
Split the process changed over time.
The Listeria monocytogenes based on double layer culture base of gainedListeria monocytogenes
The growth course schematic diagrames of the unicellular fissions of ATCC13932 is as shown in Fig. 2 as can be seen from Figure 2 food-borne pathogens first
Secondary splitting time relatively after several times splitting time it is long, be indicated above cell average division time with division number of times increase and gradually
Reduce, it is in increased trend within a certain period of time to illustrate cell splitting rate, it is observed with traditional single cells grown flowing groove
Unicellular division is contrasted, and is indicated the inventive method guarantee parent cell and is grown with daughter cell under concurrent conditions and is divided
Split, rather than the only independently growth division of observation parent cell, more conform to the growth course of individual cells in real medium.
Using the time as abscissa, sum is generated with the number of cells after fission at random and drawn for ordinate, single increasing of gained
ListeriaListeria monocytogenesCell number after the unicellular fissions of ATCC13932 changes over time situation
Schematic diagram is as shown in figure 3, from figure 3, it can be seen that being fitted obtained random growth schematic diagram by the data meets microorganism life
Long basic law, data can be provided for the structure of microbial single-cell growth model by being indicated above the inventive method
Source.
Embodiment 2
A kind of food-borne pathogens are Listeria monocytogenesListeria monocytogenesThe unicellular individuals of ATCC13932
The microscopic observation method of growth course, i.e., in Listeria monocytogenesListeria monocytogenesCovered on ATCC13932
Adapt to Listeria monocytogenesListeria monocytogenesThe solid medium of ATCC13932 growths, then with just putting type
Or inversion type microscope is observed Listeria monocytogenesListeria monocytogenesThe unicellular individuals of ATCC13932
Growth course；
Described applicable Listeria monocytogenesListeria monocytogenesATCC13932 growths solid culture used
Base is TSA-YE culture mediums；
Described TSA-YE culture mediums, its raw material composition, by every liter calculating, by 17g tryptones, 3g multivalence peptone, 6g yeast extracts,
5g sodium chloride, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, the water composition of 15g agar and surplus；
Above-mentioned TSA-YE culture mediums are by comprising the following steps that method is prepared from：
With reference to GB 4789.30-2016：Tryptone, multivalence peptone, yeast extract, sodium chloride, phosphoric acid hydrogen are sequentially added in a reservoir
Dipotassium, glucose and agar, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved, and pH is to 7.2 ± 0.2 for regulation,
Packing, 121 DEG C of autoclaving 15min are standby.
A kind of above-mentioned Listeria monocytogenesListeria monocytogenesThe unicellular individual growths of ATCC13932
The microscopic observation method of process, specifically includes following steps：
（1）, TSA-YE culture mediums, the preparation of TSB-YE culture mediums
1., the preparation of TSA-YE culture mediums
With reference to GB 4789.30-2016：Tryptone, multivalence peptone, yeast extract, sodium chloride, phosphoric acid hydrogen are sequentially added in a reservoir
Dipotassium, glucose and agar, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved, and pH is to 7.2 ± 0.2 for regulation,
Then packing, 121 DEG C of autoclaving 15min are put in standby in 50 DEG C of insulating box；
2., dilution increases the preparation of the TSB-YE culture mediums used in the Listeria monocytogenes bacteria suspension to be measured after bacterium
Above-mentioned TSB-YE culture mediums are by comprising the following steps that method is prepared from：
Sequentially add 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g chlorinations in a reservoir with reference to GB 4789.30-2016
Sodium, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, then add water and are settled to 1 liter, and then heating stirring adjusts pH to being completely dissolved
To 7.2 ± 0.2, packing, 121 DEG C of autoclaving 15min produce TSB-YE culture mediums, are then put in standby on superclean bench
With；
（2）, Listeria monocytogenes after dilutionListeria monocytogenesThe preparation of ATCC13932 bacteria suspensions
Take and increase the Listeria monocytogenes to be measured after bacteriumListeria monocytogenesATCC13932 bacteria suspensions, are used
TSB-YE culture mediums are diluted to Listeria monocytogenesListeria monocytogenesATCC13932 cell concentrations reach
To 3cfu/mL, (CFU (Colony-Forming Units, CFU) produces the Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacteria suspensions, wherein cfu/mL refer to single increasing Li Si after every milliliter of dilution
Special bacteriumListeria monocytogenesThe unicellular Listeria monocytogenes contained in ATCC13932 bacteria suspensionsListeria monocytogenesATCC13932 total plate count；
（3）, based on solid medium be used for observe Listeria monocytogenesListeria monocytogenes ATCC13932
The preparation of the sample of unicellular individual growth process
First, 20 μ L steps are taken（2）Listeria monocytogenes after the dilution of gainedListeria monocytogenes
ATCC13932 bacteria suspensions are into sterile 35mm Glass bottom culture dish, and slow shaking by swirling, make the Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacteria suspensions are paved in the bottom of Glass bottom culture dish, stand 5min；
Then, 100 μ L steps are drawn（1）TSA-YE culture mediums after sterilizing, are slightly cooled to after room temperature and are added to Glass bottom culture dish
Listeria monocytogenes after middle dilutionListeria monocytogenesOn ATCC13932 bacteria suspensions, stand until TSA-YE
Culture medium solidifies completely, then covers the upper lid of Glass bottom culture dish and seals, produce based on solid medium be used for observe list
Increase ListeriaListeria monocytogenesThe sample of the unicellular individual growth processes of ATCC13932；
（4）, microscope is observed
Using type or the microscopical 100 times of object lens of inversion type are just put, resolution ratio, field luminance, focal length are adjusted, from Glass bottom culture dish
The observation window of bottom up be observed, until finding step（3）Being used for based on solid medium of gained observes single increasing
ListeriaListeria monocytogenesUnicellular in the sample of the unicellular individual growth processes of ATCC13932
Body, records Listeria monocytogenesListeria monocytogenesATCC13932 unicellular individual cells division situation and its
The situation that Tom Clancy Splinter Cell is changed over time.
In summary, the microscopic observation method of the unicellular individual growth process of a kind of food-borne pathogens of the invention, by
The unicellular individual of food-borne pathogens is fixed on culture medium in by solid medium, therefore, food-borne cause is can observe
The unicellular overall process continuously divided of germ, and due to the presence of solid medium, observation process is not enough by nutriment
And disturb.
Above said content is only the basic explanation under present inventive concept, and according to appointing that technical scheme is done
What equivalent transformation, all should belong to protection scope of the present invention.
Claims (5)
1. a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens, it is characterised in that described food-borne
The microscopic observation method of the unicellular individual growth process of pathogenic bacteria, i.e., covering adapts to food-borne pathogens on food-borne pathogens
The solid medium or double layer culture base of growth, are then observed the unicellular individual of food-borne pathogens with microscope again
Growth course；
From top to bottom described covering double layer culture base, i.e., be followed successively by solid medium, fluid nutrient medium；
When covering adapts to the solid medium of food-borne pathogenic bacteria growing on food-borne pathogens, microscope used is just
Put type or inversion type microscope；
When covering adapts to the double layer culture base of food-borne pathogenic bacteria growing on food-borne pathogens, microscope used
For inversion type microscope.
2. a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens as claimed in claim 1, it is special
Levy and be that described food-borne pathogens are Listeria monocytogenesListeria monocytogenes ATCC13932。
3. a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens as claimed in claim 2, it is special
Levy and be described adaptation Listeria monocytogenesListeria monocytogenesThe solid medium of ATCC13932 growths
For TSA-YE culture mediums, its raw material composition, by every liter of calculating, by 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g chlorinations
Sodium, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, the water composition of 15g agar and surplus；
Above-mentioned TSA-YE culture mediums are by comprising the following steps that method is prepared from：
Tryptone, multivalence peptone, yeast extract, sodium chloride, dipotassium hydrogen phosphate, glucose and agar are sequentially added in a reservoir, so
After add water and be settled to 1 liter, then heating stirring is to being completely dissolved, and adjusts pH to 7.2 ± 0.2,121 DEG C of autoclaving 15min, standby
With；
Described adaptation Listeria monocytogenesListeria monocytogenes ATCC13932 growth fluid nutrient medium be
TSB-YE culture mediums, its raw material composition, by every liter calculating, by 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g sodium chloride,
The water composition of 2.5g dipotassium hydrogen phosphates, 2.5g glucose and surplus；
Above-mentioned TSB-YE culture mediums are by comprising the following steps that method is prepared from：
Tryptone, multivalence peptone, yeast extract, sodium chloride, dipotassium hydrogen phosphate and glucose are sequentially added in a reservoir, are then added water
1 liter is settled to, then heating stirring is to being completely dissolved, with regulation pH to 7.2 ± 0.2,121 DEG C of autoclaving 15min are standby.
4. a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens as claimed in claim 3, it is special
Levy and be to comprise the following steps that：
（1）, TSA-YE culture mediums, the preparation of TSB-YE culture mediums
The preparation of described TSA-YE culture mediums, i.e., add 17g tryptones, 3g multivalence peptone, 6g yeast extracts, 5g successively in a reservoir
Sodium chloride, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, 15g agar, then add water and are settled to 1 liter, and then heating stirring is to complete
Dissolving, adjusts pH to 7.2 ± 0.2,121 DEG C of autoclaving 15min, produces TSA-YE culture mediums；
The preparation of described TSB-YE culture mediums, i.e., sequentially add in a reservoir 17g tryptones, 3g multivalence peptone, 6g yeast extracts,
5g sodium chloride, 2.5g dipotassium hydrogen phosphates, 2.5g glucose, then add water and are settled to 1 liter, and then heating stirring is to being completely dissolved,
PH to 7.2 ± 0.2,121 DEG C of autoclaving 15min are adjusted, TSB-YE culture mediums are produced；
（2）, Listeria monocytogenes after dilutionListeria monocytogenesThe preparation of ATCC13932 bacteria suspensions
Take and increase the Listeria monocytogenes to be measured after bacteriumListeria monocytogenesATCC13932 bacteria suspensions, it is used and walked
Suddenly（1）The TSB-YE culture mediums prepared are diluted to Listeria monocytogenesListeria monocytogenes
ATCC13932 cell concentrations reach 3cfu/mL, the Listeria monocytogenes after must dilutingListeria monocytogenes
ATCC13932 bacteria suspensions, wherein cfu/mL refer to the Listeria monocytogenes after every milliliter of dilutionListeria monocytogenesThe Listeria monocytogenes contained in ATCC13932 bacteria suspensionsListeria monocytogenes
ATCC13932 total plate count；
（3）, being used for based on solid medium or double layer culture base observe Listeria monocytogenesListeria monocytogenesThe preparation of the sample of the unicellular individual growth processes of ATCC13932
First, 20 μ L steps are taken（2）Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacterium
Suspension is into sterile 35mm Glass bottom culture dish, and slow shaking by swirling, makes the Listeria monocytogenes after dilutionListeria monocytogenesATCC13932 bacteria suspensions are laid in the bottom of Glass bottom culture dish, stand 5min；
Then 100 μ L steps are drawn（1）After TSA-YE culture mediums after sterilizing are slightly cooled down, it is added to after being diluted in Glass bottom culture dish
Listeria monocytogenesListeria monocytogenesOn ATCC13932 bacteria suspensions, stand until TSA-YE culture mediums are complete
Full solidification, then covers the upper lid of Glass bottom culture dish, and produce is used to observe Listeria monocytogenes based on solid mediumListeria monocytogenesThe sample of the unicellular individual growth processes of ATCC13932；
Or 100 μ L steps are drawn first（1）After TSA-YE culture mediums after sterilizing are slightly cooled down, it is added to dilute in Glass bottom culture dish
Listeria monocytogenes after releasingListeria monocytogenesOn ATCC13932 bacteria suspensions, stand until TSA-YE cultures
Base is solidified completely, and 100-200 μ L steps are then drawn again（1）TSB-YE culture mediums after sterilizing, are covered in after above-mentioned solidification
On TSA-YE culture mediums, then cover the upper lid of Glass bottom culture dish, produce based on double layer culture base be used for observe list
Increase ListeriaListeria monocytogenesThe sample of the unicellular individual growth processes of ATCC13932；
（4）, microscope is observed
Using type or the microscopical 100 times of object lens of inversion type are just put, the single increasing corresponded in the bottom of Glass bottom culture dish after dilution
ListeriaListeria monocytogenesAt the observation window of ATCC13932 bacteria suspensions, by adjusting resolution ratio, the visual field
Brightness, focal length, find step（3）Being used for based on solid medium of gained observes Listeria monocytogenesListeria monocytogenesUnicellular individual in the sample of the unicellular individual growth processes of ATCC13932, is then observed list
Increase the process that the single celled fission process of Listeria and cell division are changed over time；
Or the microscopical 100 times of object lens of inversion type are used, the single increasing Liszt corresponded in the bottom of Glass bottom culture dish after dilution
BacteriumListeria monocytogenesAt the observation window of ATCC13932 bacteria suspensions, by adjusting resolution ratio, field luminance, Jiao
Away from finding step（3）Being used for based on double layer culture base of gained observes Listeria monocytogenesListeria monocytogenesUnicellular individual in the sample of the unicellular individual growth processes of ATCC13932, is then observed list
Increase the process that the single celled fission process of Listeria and cell division are changed over time.
5. a kind of microscopic observation method of the unicellular individual growth process of food-borne pathogens as claimed in claim 4, it is special
Levy and be that type microscope of just putting used is BX41TF-5 type biomicroscopes；
Inversion type microscope used is TS100 inversion type biomicroscopes.
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Citations (3)
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|---|---|---|---|---|
| CN1940085A (en) * | 2006-09-28 | 2007-04-04 | 东北林业大学 | Double-layer planar accounting method of bifidobacterium etc. strict anacerobe |
| WO2016060301A1 (en) * | 2014-10-16 | 2016-04-21 | 주식회사 퀀타매트릭스 | Novel biological activity testing structure for tracking single cell, using gelling agents |
| CN106498023A (en) * | 2016-09-21 | 2017-03-15 | 广东达元绿洲食品安全科技股份有限公司 | A kind of Listeria monocytogenes chromogenic culture medium and test piece |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1940085A (en) * | 2006-09-28 | 2007-04-04 | 东北林业大学 | Double-layer planar accounting method of bifidobacterium etc. strict anacerobe |
| WO2016060301A1 (en) * | 2014-10-16 | 2016-04-21 | 주식회사 퀀타매트릭스 | Novel biological activity testing structure for tracking single cell, using gelling agents |
| CN106498023A (en) * | 2016-09-21 | 2017-03-15 | 广东达元绿洲食品安全科技股份有限公司 | A kind of Listeria monocytogenes chromogenic culture medium and test piece |
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